The present invention relates generally to a method of treating viral infections, and more specifically to a method of treating rhinoviral infections.
Acute afebrile upper respiratory disease, often grouped clinically as xe2x80x9cthe common cold,xe2x80x9d is one of the most frequent afflictions of humans. Although the disease is typically not life-threatening, it causes much discomfort as well as the loss of over 200 million man-days of work and school each year in the United States alone. B. D. Davis et al., Microbiology at 1114 (Third Edition, 1980). Since 1956, when viruses from patients suffering with the common cold were first isolated, over 113 immunologically distinct, yet biologically related viruses have been isolated. Id. at 1115. The chemical and physical characteristics of these viruses led to their classification as members of the picornavirus family, and more specifically into the genus known as the Rhinoviridiae, or the rhinoviruses. Rhinoviruses have been characterized as small (17-30 nm in diameter), non-enveloped, RNA-containing virions that are cubic in structure and resistant to ether treatment. It is now thought that rhinoviral infection is the cause of approximately 40 percent of all respiratory illnesses in children and adults. V. Knight, Common Viral Respiratory Illnesses, in Harrison""s Principles of Internal Medicine, Eighth Edition at 988-990 (G. W. Thorn et al. eds., 1977).
Although several methods have been proposed as treatments for rhinoviral infection, including vaccination, air sterilization, and the administration of various pharmaceuticals, none of these methods has proved to be entirely effective. Problems that hinder the development of useful rhinovirus vaccines include the short duration of natural immunity to specific infecting types, the large number of different antigenic types of rhinovirus, and the variation of types present in a community from one year to the next. J. L. Melnick et al., McGraw-Hill Encyclopedia of Science and Technology, Seventh Edition 15, 454 (1992). Current methods of treatment are limited to the amelioration of symptoms, and often take the form of antihistamines and other drugs. There is therefore a desire to develop a method of treating rhinoviral infections that is effective, safe and practical.
In view of the foregoing, it is a first object of the present invention to provide a method for effectively and practically treating a rhinoviral infection.
A second object of the invention is to provide pharmaceutical formulations useful in the treatment of rhinoviral infections.
It has now been discovered that 2xe2x80x2-5xe2x80x2 oligoadenylates (2xe2x80x2,5xe2x80x2-A) and their analogs are surprisingly effective in the treatment of rhinoviral infections. Accordingly, a first aspect of the present invention is a method of treating a rhinoviral infection in a subject in need of such treatment. The method comprises administering to the subject a compound of Formula I: 
wherein n is 0, 1, 2, or 3;
each R1 and R2 is independently selected from the group consisting of hydrogen, halogen, and N3;
each R5 is independently hydrogen or xe2x80x94OH;
each R6 is independently hydrogen or xe2x80x94OH;
R7 is Oxe2x88x92 or xe2x80x94OH;
R9 is hydrogen or xe2x80x94OH, with the proviso that
when R9 is xe2x80x94OH, R5 is hydrogen;
Y is 
wherein m is 1, 2, or 3;
X is xe2x80x94CHOHCH2OH, or is selected from the group consisting of 
wherein R3 and R4 are each independently selected from the group consisting of hydrogen, xe2x80x94OH, and xe2x80x94PO4xe2x88x922, or R3 and R4 together represent a cyclophosphate; and A is selected from the group consisting of 
wherein each R1 and R2 are as provided above; and R8 is selected from the group consisting of hydrogen, cyanogen, and amido;
each Z is independently O or S; and
each Zxe2x80x2 is independently O or S;
or a pharmaceutically acceptable salt thereof, (hereinafter referred to as the xe2x80x9cactive compoundxe2x80x9d), in amount effective to treat the rhinoviral infection.
In one preferred embodiment of the invention, Y is xe2x80x94OH. In another preferred embodiment of the invention, X is 
R1 and R2 are hydrogen;
R3 is xe2x80x94OH and R4 is xe2x80x94PO4xe2x88x922, or
R3 is PO4xe2x88x922  and R4 is xe2x80x94OH, or
R3 and R4 are both xe2x80x94OH, or
R3 and R4 together represent a cyclophosphate;
R5 is xe2x80x94OH;
R9 is hydrogen; and
Z and Zxe2x80x2 are both O.
A second aspect of the present invention is a pharmaceutical formulation for combatting rhinoviral infection comprising, in combination with a pharmaceutically acceptable carrier, an active compound as described above.
A third aspect of the present invention is the use of an active compound as disclosed herein for the manufacture of a medicament useful in carrying out a therapeutic method of treatment as given above.
As noted above, the methods of the present invention are useful for treating infections caused by rhinoviruses. In general, rhinovirus infections (the xe2x80x9cconditionxe2x80x9d) are characterized by nasal obstruction and discharge, sneezing, moderate sore throat and mild constitutional reaction, usually without fever. In young children, these symptoms may be further aggravated by cough, croup, and pneumonia. The methods of the present invention are useful for treating these conditions in that they inhibit the onset, growth, or spread of the condition, cause regression of the condition, cure the condition, or otherwise improve the general well-being of a subject afflicted with, or at risk of contracting the condition.
Subjects to be treated by the methods of the present invention are typically human subjects, although the methods of the present invention may be useful with any suitable subject known to those skilled in the art including mammals (e.g., horses, dogs, cats) for veterinary purposes. For example, the methods may be particularly useful in treating rhinoviral infections in performing or racing animals (e.g., greyhounds, racing horses or horses used in equestrian competitions, and circus animals).
As used herein, the term xe2x80x9crhinovirusxe2x80x9d refers to viruses which are members of the picornavirus family, and more specifically to the members of the viral genus Rhinoviridiae. The rhinovirus virion is characterized as containing single stranded RNA, having a non-enveloped capsid and having cubic capsid symmetry. See Knight, supra, at 988. Rhinoviruses are sharply distinguished from other picornaviruses in that they are inactivated at low pH, maintain their infectivity at 50xc2x0 C., and have higher buoyant density in CsCl. See Davis et al., supra, at 1115.
As noted above, over 113 immunologically distinct rhinoviruses (RV) have been isolated, with each individual serotype or xe2x80x9ctypexe2x80x9d being designated by a numeral. Accordingly, rhinovirus type 1 is denoted RV-1; rhinovirus type 1, sub-type B is denoted RV-1B, etc. The present invention is useful in combatting a rhinoviral infection caused by any rhinovirus type. In a preferred embodiment, the method of the present invention is useful in combatting a rhinoviral infection caused by a rhinovirus type selected from the group consisting of rhinovirus type 1 (RV-1, including subtypes A (RV-1A) and B (RV-1B)), rhinovirus type 2 (RV-2), rhinovirus type 14 (RV-14), and rhinovirus type 39 (RV-39).
Active compounds of the present invention are, in general, 2xe2x80x2-5xe2x80x2 oligoadenylates and their analogs which have anti-rhinoviral activity. In particular, active compounds of the present invention are of Formula I, as set forth above.
In one particularly preferred embodiment of the invention, the Y group of the above Formula I is xe2x80x94OH. In another particularly preferred embodiment of the invention, X is 
R1 and R2 are hydrogen;
R3 is xe2x80x94OH and R4 is PO4xe2x88x922; or
R3 is PO4xe2x88x922  and R4 is xe2x80x94OH; or
R3 and R4 are both xe2x80x94OH; or
R3 and R4 together represent a cyclophosphate;
R5 is xe2x80x94OH;
R9 is hydrogen; and
Z and Zxe2x80x2 are both O.
2xe2x80x2-5xe2x80x2 oligoadenylate compounds and their analogs are known. Exemplary compounds include 2xe2x80x2-5xe2x80x2 oligoadenylate, the 2xe2x80x2,5xe2x80x2-oligoadenylate- 2xe2x80x2,3xe2x80x2 cyclophosphates disclosed in German Patent DE 42 11 237 A1 to Budowsky, et al.; the 2xe2x80x2,5xe2x80x2-oligoadenylate phosphate-containing derivatives disclosed in German Patent DE 42 11 238 C2 to Budowsky et al.; the 2xe2x80x2- and 8-azido(2xe2x80x2-5xe2x80x2)oligoadenylates disclosed in U.S. Pat. No. 4,990,498 to Suhadolnik; the 2xe2x80x2-5xe2x80x2-phosphorothioate oligoadenylates in disclosed in U.S. Pat. No. 5,188,897 to Suhadolnik et al.; the 2xe2x80x2-5xe2x80x2 riboadenylate-morpholinoadenylate oligonucleotides disclosed in U.S. Pat. No. 4,515,781 to Torrence et al.; the (2xe2x80x2-5xe2x80x2)-oligo(3xe2x80x2-deoxyadenylates) and derivatives thereof disclosed in U.S. Pat. No. 4,464,359 to Suhadolnik et al.; the 2xe2x80x2-5xe2x80x2 oliogadenylate cordycepin analogs as disclosed in U.S. Pat. No. 4,859,768 to Suhadolnik et al.; the 2xe2x80x2-5xe2x80x2 oligoxyloadenylates disclosed in U.S. Pat. No. 4,476,301 to Imbach et al.; and the 8-azaadenosine, sagivamycin, toyocamycin, tubericidine, and 8-bromo-adenosine analogs of 2xe2x80x2-5xe2x80x2 oligoadenylates as disclosed in U.S. Pat. No. 4,981,957 to Lebleu et al. (applicants specifically intend that the disclosure of these and all other patent references cited herein be incorporated by reference herein in their entirety).
Specific compounds useful in the practice of the present invention include, but are not limited to, 2xe2x80x2,3xe2x80x2-cyclophosphateadenylyl(2xe2x80x2,5xe2x80x2)adenylyl-(2xe2x80x2,5xe2x80x2)adenosine (Papirine AIII), 2xe2x80x2,3xe2x80x2-cyclophosphateadenylyl(2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)adenylyl-(2xe2x80x2,5xe2x80x2)adenosine (Papirine AIV), 2xe2x80x2,3xe2x80x2-cyclophosphateadenylyl(2xe2x80x2,5xe2x80x2)adenosine (Papirine AII), 2xe2x80x2-phosphateadenylyl(2xe2x80x2,5xe2x80x2)adenylyl-(2xe2x80x2,5xe2x80x2)adenosine (Papirine BIII), 3xe2x80x2-phosphateadenylyl(2xe2x80x2,5xe2x80x2)adenylyl-(2xe2x80x2,5xe2x80x2)adenosine (Papirine BIII), 2xe2x80x2-phosphateadenylyl(2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)adenylyl-(2xe2x80x2,5xe2x80x2)adenosine (Papirine BIV), 3xe2x80x2-phosphateadenylyl (2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)-adenosine (Papirine BIV), 2xe2x80x2-phosphateadenylyl(2xe2x80x2,5xe2x80x2)adenosine (Papirine BII), 3xe2x80x2-phosphateadenylyl(2xe2x80x2,5xe2x80x2)adenosine (Papirine BII), adenylyl(2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)adenosine (Papirine CIII), adenylyl(2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)adenylyl-(2xe2x80x2,5xe2x80x2)adenosine (Papirine CIV), adenylyl(2xe2x80x2,5xe2x80x2)-adenosine (Papirine CII), adenylyl(2xe2x80x2,5xe2x80x2)3xe2x80x2-deoxyadenylyl(2xe2x80x2,5xe2x80x2)3xe2x80x2-deoxyadenosine and the 5xe2x80x2 mono-, di-, and triphosphates thereof, 3xe2x80x2-deoxyadenylyl(2xe2x80x2,5xe2x80x2)31-deoxyadenosine and the 5xe2x80x2 mono-, di-, and triphosphates thereof, adenylyl(2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)tubercidin and the 5xe2x80x2 mono-, di-, and triphosphates thereof, 5xe2x80x2-O-p-methoxytrityladenylyl(2xe2x80x2,5xe2x80x2)adenylyl(2xe2x80x2,5xe2x80x2)adenosine, xyloadenylyl(2xe2x80x2,5xe2x80x2)xyloadenylyl(2xe2x80x2,5xe2x80x2)xyloadenosine, (Rp)-P-thioadenylyl-(2xe2x80x2,5xe2x80x2)-(Sp)-thioadenylyl(2xe2x80x2,5xe2x80x2)adenosine, and (Sp)-P-thioadenylyl-(2xe2x80x2,5xe2x80x2)-(Rp)-thioadenylyl(2xe2x80x2,5xe2x80x2)adenosine.
Compounds of the present invention may be prepared using chemical enzymatic synthesis methods which will be apparent to one skilled in the art. For example, 2xe2x80x2,5xe2x80x2-oligoadenylate-2xe2x80x23xe2x80x2-cyclophosphates may be produced beginning with poly(A) with irregular 2xe2x80x2,5xe2x80x2 and 3xe2x80x2,5xe2x80x2 internucleotide bonds, using the procedure of Michaelson (A. M. Michaelson, The Chemistry of the Nucleosides and Nucleotides, 418-19 (1963)), by the chemical polymerization of 2xe2x80x2 (3xe2x80x2) adenosine monophosphate. The subsequent split of the 3xe2x80x251 bonds in this polymer by ribonuclease leads to a monomer and 2,xe2x80x25 oligoadenylates of varying lengths with a mixture containing a terminal 2xe2x80x2,3xe2x80x2 cyclophosphate group. By keeping the mixture at a low pH, the terminal cyclophosphate can be opened to provide other compounds useful in the method of this invention. Additional synthetic pathways useful in providing compound useful in the invention are disclosed in U.S. Pat. No. 4,981,957 to Lebleu et al., U.S. Pat. No. 4,708,935 to Suhadolnik et al., U.S. Pat. No. 4,990,498 to Suhadolnik et al., U.S. Pat. No. 4,515,781 to Torrence et al., and U.S. Pat. No. 4,476,301 to Imbach et al.
The active compounds described herein can, as noted above, be prepared in the form of their pharmaceutically acceptable salts. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts include salts derived from bases, such as ammonium salts; alkali metal salts such as those of sodium and potassium; alkaline earth metal salts such as those of calcium and magnesium; and salts with organic bases such as triethylamine, dicyclohexylamine, and the like.
In order to enhance the intracellular transport of the active compounds, the compounds can also be conjugated to macromolecular carriers (e.g., poly(L-lysine)), as described in U.S. Pat. No. 5,405,939 to Suhadolnik et al.
The therapeutically effective dosage of any specific compound, the use of which is in the scope of present invention, will vary somewhat from compound to compound, patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 100 ng/kg to about 1 mg/kg will have therapeutic efficacy, with a dosage of from about 10 xcexcg/kg to 100 xcexcg/kg being preferred, with all weights being calculated based upon the weight of the active base, including the cases where a salt is employed. A dosage from about 10 xcexcg/kg to about 10 mg/kg may be employed for oral administration, with a dosage from about 1 mg/kg to 10 mg/kg being preferred. Treatment can be administered once daily or several times per day for a period of 1 to 10 days or until the rhinoviral infection is essentially controlled. Lower doses given less frequently can be used to prevent or reduce the incidence of recurrence of the infection.
Depending on the solubility of the particular formulation of active compound administered, the daily dose may be divided among one or several unit dose administrations. The dose may be a single unit dose, which may, for example, be administered several times a week or from 1 to 3 times a day. Treatments may continue week to week on a chronic basis as necessary (i.e., the active agent can be administered repeatedly). Administration of the active compounds may be carried out therapeutically or prophylactically, but preferably the compounds are administered therapeutically, either before symptoms of the rhinoviral infection have appeared, or at a time when such symptoms are first appearing.
In accordance with the present method, an active compound as described herein may be prepared as a formulation suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation, rectal, topical (including buccal, sublingual, dermal and intraocular), and transdermal administration.
In the manufacture of a medicament according to the invention (a xe2x80x9cformulationxe2x80x9d), active agents or the pharmaceutically acceptable salts thereof (the xe2x80x9cactive compoundxe2x80x9d) are typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier may be solid or liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.05% to 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention (e.g. the formulation may contain one or more additional antiviral agents as noted above), which formulations may be prepared by any of the well-known techniques if pharmacy consisting essentially of admixing the components, including one or more accessory therapeutic ingredients.
In addition to the active compounds or their salts, the pharmaceutical compositions may contain other additives, such as pH adjusting additives. In particular, useful pH adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the compositions may contain microbial preservatives. Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. Of course, as indicated, the pharmaceutical compositions of the present invention may be lyophilized using techniques well known in the art.
As used in the present specification, the term xe2x80x9cwater-solublexe2x80x9d is meant to define any composition which is soluble in water in an amount of about 50 mg/mL, or greater. Also, as used in the present specification, the term xe2x80x9cwater-insolublexe2x80x9d is meant to define any composition which has solubility in water of less than about 1 mg/mL. compositions that are soluble in water in an amount from about 1 mg/mL to about 60 mg/mL are defined as xe2x80x9cpartially soluble.xe2x80x9d For certain applications, water soluble compounds or salts may be desirable, whereas for other applications water-insoluble compounds or salts likewise may be desirable.
Pharmaceutical compositions may be prepared from the water-insoluble active compounds, or salts thereof, such as aqueous base emulsions. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the active compound or salt thereof. Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.
Further, the present invention provides liposomal formulations of the active compounds and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the active compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.
Of course, the liposomal formulations containing the active compounds or salts thereof, may be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder. Preferably, formulations for oral administration may include enteric coatings known in the art to prevent degradation of the formulation in the stomach and provide release of the drug in the small intestine. Also, the dosage of the formulation may be raised slightly to overcome any digestion of the active compound in the gastrointestinal (GI) tract. Alternatively, a xe2x80x9cdiverting compoundxe2x80x9d (e.g., a 3xe2x80x2,5xe2x80x2-oligoadenylate compound) may be administered in conjunction with the active compound in order to provide competing substrates for such GI enzymes.
Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection preparations of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may include anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The formulations may be presented in unit/dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Pharmaceutical formulations are also provided which are suitable for administration as an aerosol, by inhalation. These formulations comprise a solution or suspension of the desired active compound or a salt thereof or a plurality of solid particles of the compound or salt. The desired formulation may be placed in a small chamber and nebulized. Nebulization may be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the compounds or salts. The liquid droplets or solid particles should have a particle size in the range of about 0.5 to about 5 microns. The solid particles can be obtained by processing the solid active compound, or a salt thereof, in any appropriate manner known in the art, such as by micronization. Most preferably, the size of the solid particles or droplets will be from about 1 to about 2 microns. In this respect, commercial nebulizers are available to achieve this purpose.
Preferably, when the pharmaceutical formulation suitable for administration as an aerosol is in the form of a liquid, the formulation will comprise a water-soluble active compound of the present invention or a salt thereof, in a carrier which comprises water. A surfactant may be present which lowers the surface tension of the formulation sufficiently to result in the formation of droplets within the desired size range when subjected to nebulization.
Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Pharmaceutical Research 3, 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.